DVT, whether accompanied by pulmonary embolism or not, is a potentially serious complication of many surgical and medical conditions which requires early diagnosis as a basis for prompt and appropriate treatment in order to counteract serious sequelae. The incidence of DVT is appreciable under traumatic conditions, such as those associated with major surgery or bone fracture (particularly of the neck of femur). Furthermore, it is well recognized that any condition of enforced immobility, such as advanced age, obesity, neoplasm and cardiac failure or infarction enhances the risk of developing DVT.
The correct clinical diagnosis of DVT is impeded by the fact that clinical symptoms and signs thereof tend to be rather unspecific and often unreliable. Thus the clinical diagnosis of DVT based on such symptoms as venous distension, pain, tenderness and swelling of the affected limb with increased skin temperature has often proved to give false positive results. Contrarywise, it has also been recognized that even extensive DVT may occur in the absence of conspicuous physical signs if the main veins are not completely occluded and that, in fact, a substantial number of patients with DVT do not show clinical evidence thereof.
The inadequacy of clinical examination has led to the search for objective diagnostic tests for the detection of DVT. Over the years a variety of such tests, ranging from X-ray phlebography over methods based on monitoring blood flow (for example by Doppler ultrasound technique or impedence plethysmography) to localization of thrombi by means of radiopharmaceuticals, have become available to the medical profession. The latter group of tests makes use of radiopharmaceuticals having the ability to accumulate in fibrinous clots, thereby enabling their dectection, for example by scintigraphic scanning procedures. Exemplary of such radiopharmaceuticals are radioiodinated fibrinogen and technetium-99m labelled plasmin. Whereas the labelled fibrinogen is incorporated rather slowly into the developing thrombus, extraneous labelled plasmin has been found to accumulate rapidly even in established fibrinous clots.
Mammalian blood contains an enzymatic system which is called the fibrinolytic enzyme system. Under normal conditions a dynamic equilibrium is maintained where fibrin deposits are dissolved by local activation of the fibrinolytic enzyme system. Endogenous activators, for example the so called plasminogen tissue activator, convert the circulating plasminogen into the fibrinolytically active enzyme plasmin which has a high affinity for fibrin. Among exogenous activators of the fibrinolytic enzyme system mention may be made of streptokinase which is generated by certain strains of haemolytic streptococci, urokinase which is recoverable from human urine or produced in tissue culture, and trypsin.
Owing to its fibrinolytic effect infusion of exogenous plasmin has been used as an alternative to other methods in thrombolytic therapy. To satisfy the requirements for such therapeutic purposes plasmin may be prepared from purified plasminogen by activation with streptokinase or, more frequently, with urokinase or trypsin.
For diagnostic purposes the detection of fibrin containing thrombi may be achieved by injection of small amounts of radiolabelled plasmin. Due to the rapidly established association of radiolabelled plasmin with fibrinous clots, particularly in comparison with such agents as radioiodinated fibrinogen, the technetium-99m plasmin test has been shown to be a highly sensitive and versatile procedure which is well adapted for relatively fast routine screening of patients for developing as well as established DVT (vide British Journal of Radiology, vol. 53 (1980) p. 673, J. M. Deacon et al.).
The same reference describes a technetium-99m plasmin kit in which the plasmin is admixed with sodium chloride together with stannous chloride as the .sup.99 Tc.sup.m -pertechnetate reducing agent. This method for labelling plasmin with technetium-99m was first described by R. B. R. Persson and L. Darte (Int. Journal of Applied Radiation and Isotopes, vol. 28 (1977) p. 97).
The technetium-99m plasmin kit, comprising a lyophilized mixture of plasmin and stannous chloride, has been available for some time to hospitals engaged in trial programmes directed to the evaluation of the technetium-99m-plasmin test as a screening method for diagnosing DVT. The kit has been supplied by NOVO INDUSTRI A/S, Copenhagen under the name "LYSOFIBRIN kit for .sup.99 Tc.sup.m -labelling" (LYSOFIBRIN is registered trade mark for highly purified porcine plasmin).
It has been found, however, that the plasmin incorporated into this kit is not sufficiently stable for its intended use unless the kit is permanently stored at a temperature not exceeding -20.degree. C. Thus at the ordinary refigerator temperature of 5.degree. C. inactivation of plasmin proceeds at a rate of approximately 5 per cent per month. Since retention of proteolytic activity of plasmin is believed to be a prerequisite for its affinity to fibrin, which property confers specificity on the radiolabelled plasmin for thrombi, the instability problem should be eliminated before the kit is made available for general clinical use.
It is an object of the present invention to devise plasmin compositions conditioned for technetium-99m labelling which are devoid of the shortcomings pertaining to the composition described hereinbefore.
It is a further object of this invention to provide a process for preparing such plasmin compositions in which the plasmin is stabilized to the extent that the composition is suited for its intended use even after storage for extended periods of time under ambient conditions.
The attainment of these objects are based on the discovery that certain polyhydroxy compounds, when incorporated into the composition, elicit a pronounced stabilizing effect on the plasmin therein without, however, interfering substantially neither with the yield of technetium-99m plasmin obtained in a subsequent labelling process conducted under standardized conditions nor with the radiochemical purity of the resulting radiopharmaceutical.
Glycerol is used as a stabilizer of plasmin in aqueous solution, usually at a concentration of about 50 percent of the solution, i.e. vastly in excess of plasmin on a weight basis. However, liquid polyhydroxy compounds are not contemplated for the compositions of this invention due to the mandatory presence therein of a strongly reducing agent which may be susceptible to liquid phase deterioration. Hence, the stabilized plasmin compositions of this invention are in solid form. Furthermore, it may be mentioned that the stabilizers usable for labelling purposes should generally be physiologically acceptable, should not themselves be labelled under labelling conditions preferred herein, and should not interfere with the labelling of plasmin.
Polyhydroxy compounds, such as sugars and sugar alcohols, are known as stabilizers of proteolytic enzymes, particularly in liquid formulations thereof, in which inactivation is believed to be caused mainly by enzyme autodigestion. However, the problem facing the inventors in the present case is a different one, firstly because the plasmin compositions of the present invention are solid, and secondly because they are prepared and used under acid conditions where the plasmin therein is proteolytically inactive. Therefore, inactivation must be caused by other factors than autodigestion.
As will be outlined subsequently, it has surprisingly been found that the polyhydroxy compounds devised herein prevent or at least impede the aggregation of plasmin. Since such plasmin aggregates are believed to represent initial stages of denatured plasmin, their presence in the compositions of this invention is undesirable.